Sperm quality variables of sex-sorted bull semen produced by magnetic-activated cell sorting coupled with recombinant antibodies targeting Y-chromosome-bearing sperm.

The immunological sexing method using antibodies offers cost-effective, high-volume production but faces challenges in terms of X-sperm purity in sexed semen. This research aimed to produce sexed bull semen using highly specific recombinant antibodies in magnetic-activated cell sorting (MACS), evaluate sperm quality and kinematic parameters, and verify the sex ratio of sperm, embryos, and live calves. Fresh semen from two Angus bulls was separated into two equal groups: conventional (CONV) semen and semen sexed using MACS with Y-scFv antibody conjugation to separate two fractions, i.e., the X-enriched and Y-enriched fractions. Then, computer assisted semen analysis and imaging flow cytometry were used to evaluate sperm motility and kinematic variables, acrosomal integrity, sperm viability, and sperm sex ratios. The results showed that sperm motility and quality did not differ between X-enriched and CONV semen. However, the Y-enriched fraction showed significantly lower sperm quality than the X-enriched fraction and CONV semen. The sperm ratio revealed that X-sperm accounted for up to 79.50% of the X-enriched fraction, while Y-sperm accounted for up to 78.56% of the Y-enriched fraction. The sex ratio of embryos was examined using in vitro fertilization. The cleavage rates using CONV and X-enriched semen were significantly higher than that using Y-enriched semen. Accordingly, 88.26% female blastocysts were obtained by using X-enriched semen, and 83.58% male blastocysts were obtained by using Y-enriched semen. In farm trials, 304 cows were subjected to AI using X-enriched and CONV semen. The pregnancy rate did not differ between the X-enriched and CONV semen groups. On the other hand, X-enriched semen generated significantly more live female calves (83.64%) than CONV semen (47.00%). The MACS sexing method significantly enhanced the X-sperm purity in sexed semen, producing high-quality sperm, a high percentage of female blastocytes, and a high percentage of live female calves.

Effect of Electroejaculation Protocols on Semen Quality and Concentrations of Testosterone, Cortisol, Malondialdehyde, and Creatine Kinase in Captive Bengal Tigers.

The Bengal tiger (Panthera tigris tigris) is critically endangered, so assisted reproductive technologies, including artificial insemination, are important conservation tools. For wild and domestic felids, electroejaculation (EE) is the most common semen collection method, with protocols optimized to obtain sufficient amounts of viable sperm for artificial insemination. However, less attention has been paid to ensuring animal wellbeing during the process. This study examined the effects of three EE protocols (Low, 2-5 volts; Medium, 3-6 volts; High, 4-7 volts) on semen quality, testicular size, serum testosterone, creatine kinase (CK), and malondialdehyde (MDA) concentrations, and serum cortisol as a proxy for stress. Blood samples were collected before, during, and after each EE series. Seminal plasma pH, and sperm motility, viability, and morphology were evaluated after each procedure. Seminal plasma and sperm pellet MDA concentrations were also determined. Primary sperm abnormalities and seminal plasma MDA were higher in the Low compared to Medium and High voltage groups (p < 0.05). Serum CK in the High voltage group increased during the EE series (p < 0.05), suggesting the potential for muscle damage. However, no significant changes were observed for serum cortisol, testosterone, or MDA concentrations. Results suggest the Medium voltage protocol produced good quality samples at lower voltages than the High protocol with no negative effect on muscle function, which might be better for animal welfare.

High-Efficiency Bovine Sperm Sexing Used Magnetic-Activated Cell Sorting by Coupling scFv Antibodies Specific to Y-Chromosome-Bearing Sperm on Magnetic Microbeads.

Sperm sexing technique is favored in the dairy industry. This research focuses on the efficiency of bovine sperm sexing using magnetic-activated cell sorting (MACS) by scFv antibody against Y-chromosome-bearing sperm (Y-scFv) coupled to magnetic microbeads and its effects on kinematic variables, sperm quality, and X/Y-sperm ratio. In this study, the optimal concentration of Y-scFv antibody coupling to the surface of magnetic microbeads was 2-4 mg/mL. PY-microbeads revealed significantly enriched Y-chromosome-bearing sperm (Y-sperm) in the eluted fraction (78.01-81.43%) and X-chromosome-bearing sperm (X-sperm) in the supernatant fraction (79.04-82.65%). The quality of frozen-thawed sexed sperm was analyzed by CASA and imaging flow cytometer, which showed that PY-microbeads did not have a negative effect on X-sperm motility, viability, or acrosome integrity. However, sexed Y-sperm had significantly decreased motility and viability. The X/Y-sperm ratio was determined using an imaging flow cytometer and real-time PCR. PY-microbeads produced sperm with up to 82.65% X-sperm in the X-enriched fraction and up to 81.43% Y-sperm in the Y-enriched fraction. Bovine sperm sexing by PY-microbeads showed high efficiency in separating Y-sperm from X-sperm and acceptable sperm quality. This initial technique is feasible for bovine sperm sexing, which increases the number of heifers in dairy herds while lowering production expenses.

Freezability biomarkers in the epididymal spermatozoa of swamp buffalo.

As an alternative to ejaculated semen, epididymal spermatozoa (ES) can be recovered and cryopreserved for use in artificial insemination and other assisted reproductive technologies. However, variabilities in the sperm response to freeze-thaw procedures challenge its inherent value. Therefore, the present study aims to clarify the freezability phenomena in the swamp buffalo ES, where pieces of literature do not abound. Here, we isolated ES from swamp buffaloes using abattoir-derived, post-mortem caudal epididymis by slicing-flushing technique. Following cryopreservation by slow-freezing, ES samples were classified into high (HF) and low freezability (LF) based on their post-thaw total and progressive motilities. Conventional sperm parameters and proteins of interest, such as glutathione S-transferase Mu 3 (GSTM3) and ATP synthase beta subunit (ATP1B1), were assessed and compared between HF and LF. Computer-assisted sperm analysis revealed that nearly all motion and kinematic parameters significantly differed among freezability groups except for wobble, linearity, and straightness. Moreover, intracellular reactive oxygen species production was evident in both HF and LF after fluorescence staining, with the latter having considerably greater malondialdehyde levels than the former. Immunohistochemical labeling demonstrated that both GSTM3 and ATP1B1 proteins were present in the ES and the epididymal tubular epithelium. Although the GSTM3 relative amounts, as analyzed through Western blot, were significantly higher in LF than HF in association with lipid peroxidation, no significant differences were observed in the case of ATP1B1. Such variations in motility, motion and kinematics, oxidative stress status, and specific sperm proteins suggest their potential utility in distinguishing freezability phenotypes in swamp buffalo ES.

Vitrification of Pronuclear Zygotes Perturbs Porcine Zygotic Genome Activation.

Zygotic genome activation (ZGA) plays an essential role in early embryonic development. Vitrification is a common assisted reproductive technology that frequently reduces the developmental competence of embryos. However, the effect of vitrification on porcine ZGA and gene expression during ZGA remains largely unclear. Here, we found that vitrification of pronuclear zygotes derived from parthenogenetic activation (PA) and in vitro fertilization (IVF) resulted in a significant reduction in the rates of 2-cell, 4-cell, and blastocysts, but did not affect the quality of blastocysts. Functional research revealed that RNA polymerase II Inhibitor (α-amanitin) treatment significantly reduced global transcriptional activity and developmental efficiency of both 4-cell and 8-cell embryos, implying an essential role of ZGA in porcine early embryonic development. Furthermore, vitrification did not affect the synthesis of nascent mRNA of 2-cell embryos, but significantly inhibited global transcriptional activity of both 4-cell and 8-cell embryos, suggesting an impaired effect of vitrification on porcine ZGA. Correspondingly, the single-cell analysis showed that vitrification caused the downregulation or upregulation expression of maternal genes in 4-cell embryos, also significantly decreased the expression of zygotic genes. Taken together, these results indicated that vitrification of pronuclear zygotes impairs porcine zygotic genome activation.

Dynamic reprogramming and function of RNA N6-methyladenosine modification during porcine early embryonic development.

N6-Methyladenosine (m6A) regulates oocyte-to-embryo transition and the reprogramming of somatic cells into induced pluripotent stem cells. However, the role of m6A methylation in porcine early embryonic development and its reprogramming characteristics in somatic cell nuclear transfer (SCNT) embryos are yet to be known. Here, we showed that m6A methylation was essential for normal early embryonic development and its aberrant reprogramming in SCNT embryos. We identified a persistent occurrence of m6A methylation in embryos between 1-cell to blastocyst stages and m6A levels abruptly increased during the morula-to-blastocyst transition. Cycloleucine (methylation inhibitor, 20 mM) treatment efficiently reduced m6A levels, significantly decreased the rates of 4-cell embryos and blastocysts, and disrupted normal lineage allocation. Moreover, cycloleucine treatment also led to higher levels in both apoptosis and autophagy in blastocysts. Furthermore, m6A levels in SCNT embryos at the 4-cell and 8-cell stages were significantly lower than that in parthenogenetic activation (PA) embryos, suggesting an abnormal reprogramming of m6A methylation in SCNT embryos. Correspondingly, expression levels of m6A writers (METTL3 and METTL14) and eraser (FTO) were apparently higher in SCNT 8-cell embryos compared with their PA counterparts. Taken together, these results indicated that aberrant nuclear transfer-mediated reprogramming of m6A methylation was involved in regulating porcine early embryonic development.

Impact of Cryopreservation on Spermatozoa Freeze-Thawed Traits and Relevance OMICS to Assess Sperm Cryo-Tolerance in Farm Animals.

Sperm cryopreservation is a powerful tool for the livestock breeding program. Several technical attempts have been made to enhance the efficiency of spermatozoa cryopreservation in different farm animal species. However, it is well-recognized that mammalian spermatozoa are susceptible to cryo-injury caused by cryopreservation processes. Moreover, the factors leading to cryo-injuries are complicated, and the cryo-damage mechanism has not been methodically explained until now, which directly influences the quality of frozen-thawed spermatozoa. Currently, the various OMICS technologies in sperm cryo-biology have been conducted, particularly proteomics and transcriptomics studies. It has contributed while exploring the molecular alterations caused by cryopreservation, identification of various freezability markers and specific proteins that could be added to semen diluents before cryopreservation to improve sperm cryo-survival. Therefore, understanding the cryo-injury mechanism of spermatozoa is essential for the optimization of current cryopreservation processes. Recently, the application of newly-emerged proteomics and transcriptomics technologies to study the effects of cryopreservation on sperm is becoming a hotspot. This review detailed an updated overview of OMICS elements involved in sperm cryo-tolerance and freeze-thawed quality. While also detailed a mechanism of sperm cryo-injury and utilizing OMICS technology that assesses the sperm freezability potential biomarkers as well as the accurate classification between the excellent and poor freezer breeding candidate.

METTL3-mediated m6A methylation negatively modulates autophagy to support porcine blastocyst development‡.

N6-methyladenosine (m6A) catalyzed by METTL3 regulates the maternal-to-zygotic transition in zebrafish and mice. However, the role and mechanism of METTL3-mediated m6A methylation in blastocyst development remains unclear. Here, we show that METTL3-mediated m6A methylation sustains porcine blastocyst development via negatively modulating autophagy. We found that reduced m6A levels triggered by METTL3 knockdown caused embryonic arrest during morula-blastocyst transition and developmental defects in trophectoderm cells. Intriguingly, overexpression of METTL3 in early embryos resulted in increased m6A levels and these embryos phenocopied METTL3 knockdown embryos. Mechanistically, METTL3 knockdown or overexpression resulted in a significant increase or decrease in expression of ATG5 (a key regulator of autophagy) and LC3 (an autophagy marker) in blastocysts, respectively. m6A modification of ATG5 mRNA mainly occurs at 3'UTR, and METTL3 knockdown enhanced ATG5 mRNA stability, suggesting that METTL3 negatively regulated autophagy in an m6A dependent manner. Furthermore, single-cell qPCR revealed that METTL3 knockdown only increased expression of LC3 and ATG5 in trophectoderm cells, indicating preferential inhibitory effects of METTL3 on autophagy activity in the trophectoderm lineage. Importantly, autophagy restoration by 3MA (an autophagy inhibitor) treatment partially rescued developmental defects of METTL3 knockdown blastocysts. Taken together, these results demonstrate that METTL3-mediated m6A methylation negatively modulates autophagy to support blastocyst development.

Histology of 24 organs from Asian elephant calves (Elephas maximus).

BACKGROUND: Elephants are the largest and heaviest living terrestrial animals, but information on their histology is still lacking. This study provides a unique insight into the elephant's organs and also provides a comparison between juvenile Asian elephants and adult Asian elephants or other species. Here we report on the histological structure of 24 organs, including the skin, brain (cerebrum, cerebellar hemisphere, vermis, thalamus, midbrain), spinal cord, sciatic nerve, striated skeletal muscle, cardiac muscle, bone (flat bone and long bone), cartilage (hyaline cartilage and fibrocartilage), heart (right atrium, right ventricle), blood vessels (aorta, pulmonary artery and caudal vena cava), trunk, trachea, lung, tongue, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), liver and pancreas, kidney, ovary, uterus (body and horn) and spleen of two juvenile Asian elephants. METHODS: Tissue sections were stained with Harris's hematoxylin and eosin Y. RESULTS: While almost all structures were similar to those of other species or adult elephants, some structures were different from other mammalian species, such as: plexiform bone was found in flat bone only; a thin trachealismuscle was observed in the trachea; and no serous or mucinous glands were found in the submucosa of the trachea. DISCUSSION: Histological information from various organs can serve as an important foundation of basal data for future microanatomical studies, and help in the diagnosis and pathogenesis in sick elephants or those with an unknown cause of death.

Intravenous transplantation of mouse embryonic stem cells attenuates demyelination in an ICR outbred mouse model of demyelinating diseases.

Induction of demyelination in the central nervous system (CNS) of experimental mice using cuprizone is widely used as an animal model for studying the pathogenesis and treatment of demyelination. However, different mouse strains used result in different pathological outcomes. Moreover, because current medicinal treatments are not always effective in multiple sclerosis patients, so the study of exogenous cell transplantation in an animal model is of great importance. The aims of the present study were to establish an alternative ICR outbred mouse model for studying demyelination and to evaluate the effects of intravenous cell transplantation in the present developed mouse model. Two sets of experiments were conducted. Firstly, ICR outbred and BALB/c inbred mice were fed with 0.2% cuprizone for 6 consecutive weeks; then demyelinating scores determined by luxol fast blue stain or immunolabeling with CNPase were evaluated. Secondly, attenuation of demyelination in ICR mice by intravenous injection of mES cells was studied. Scores for demyelination in the brains of ICR mice receiving cell injection (mES cells-injected group) and vehicle (sham-inoculated group) were assessed and compared. The results showed that cuprizone significantly induced demyelination in the cerebral cortex and corpus callosum of both ICR and BALB/c mice. Additionally, intravenous transplantation of mES cells potentially attenuated demyelination in ICR mice compared with sham-inoculated groups. The present study is among the earliest reports to describe the cuprizone-induced demyelination in ICR outbred mice. Although it remains unclear whether mES cells or trophic effects from mES cells are the cause of enhanced remyelination, the results of the present study may shed some light on exogenous cell therapy in central nervous system demyelinating diseases.

The effect of M-phase stage-dependent kinase inhibitors on inositol 1,4,5-trisphosphate receptor 1 (IP3 R1) expression and localization in pig oocytes.

At fertilization, inositol 1,4,5-trisphosphate receptor type 1 (IP3 R1) has a crucial role in Ca(2+) release in mammals. Expression levels, localization and phosphorylation of IP3 R1 are important for its function, but it still remains unclear which molecule(s) regulates IP3 R1 behavior in pig oocytes. We examined whether there was a difference in localization of IP3 R1 after in vitro or in vivo maturation of pig oocytes. In mouse oocytes, large clusters of IP3 R1 were formed in the cortex of the oocyte except in a ring-shaped band of cortex adjacent to the spindle. However, no such clusters of IP3 R1 were observed in pig oocytes and there was no difference in its localization between in vitro and in vivo matured oocytes. We next tried to clarify which factor(s) regulates IP3 R1 localization, phosphorylation and expression using M-phase stage-dependent kinase inhibitors. Our results show that treatments with roscovitine (p34(cdc2) kinase inhibitor) or U0126 (mitogen-activated protein kinase inhibitor) did not affect IP3 R1 expression or localization in pig oocytes, although the latter strongly inhibited phosphorylation. However, treatment with BI-2536, an inhibitor of polo-like kinase 1 (Plk1), dramatically decreased the expression level of IP3 R1 in pig oocytes in a dose-dependent manner. From these results, it is suggested that Plk1 is involved in the regulation of IP3 R1 expression in pig oocytes.